The Role of Mismatch-Repair
Protein MLH3 in Genomic Stability
in the Plant Arabidopsis
Howard Hughes Medical Institute
Summer Research Program
Laurel Wheeler
Mentor: Dr. John Hays
DNA Repair Systems
•One major concern for an organism’s survival
and fecundity is genomic stability
•Errors in DNA can occur during synthesis or
post replication from environmental factors (i.e.
UV radiation)
•This is a major problem because errors lead to
mutations, which can lead to a variety of
problems including cancer
•Organisms have multiple repair systems
•Direct Reversal
•Base Excision
•Recombination
•Nucleotide Excision
•Mismatch Repair
Mismatch Repair (MMR)
MMR is responsible for locating and
removing mismatched base pairs
Highly conserved throughout evolution
Increases genomic stability 100 to 1,000
fold
Lack of MMR has been linked to several
forms of cancer
Recognition of Daughter Strand
MMR telling the difference
between daughter and parent
stands is critical in repair
In prokaryotes MMR recognizes
the difference between the two
strands by looking for the
unmethylated strand
A stand is methylated shortly
following replication, thus the
unmethylated strand is the
daughter stand
Prokaryotic MMR
Mismatch is recognized
MutS binds to DNA at
mismatch site
MutL recruited by MutS
MutL acts as matchmaker
between MutS and MutH
Recognizes correct strand
because it is unmethylated
MutH creates a nick 5’ to the
unmethylated GATC site
Exonuclease degrades
section of the strand
containing mismatch
Gap in DNA is resynthesized
http://cmgm.stanford.edu/biochem201/Slides/DNA%20Repair/24%20mismatch%20r
Eukaryotic MMR
Instead of MutS, MutL, and MutH, there
are several MutS and MutL homologues
MutS and MutL homologues exist in
heterodimers
Arabidopsis thaliana
Investigation
of the MLH3
homologue
found in
Arabidopsis
plants
MLH3 ???
Unknown function of
MLH3
Use MLH3 knockout
mutants to look for
irregularities in plants
lacking this part of
MMR
Possible problems
with meiosis?
Sterility? Phenotypic
changes?
Current Experiments Underway
Look for MLH3 knockout homozygous
lines
Identify homozygotes through genotyping
Confirm through MSI assays
Genotyping the Plants
PCR using primers LBa1
and MLH3-R
PCR using primers MLH3-F
and MLH3-R
MMR Correction of Slip-Mispairing
Replication
AT
NNNATATAT ATATAT
NNNTATATA TATATATATATANNN
+2 base insertion
MMR
NNNATATATATATAT
NNNTATATATATATATATATANNN
no insertion
or deletion
MMR
NNNATATAT ATATAT
NNNTATATA TATATATATATANNN
TA
-2 base deletion
Microsatellite Instability (MSI)
• PCR was run using
primers specific for
microsatellite sites
• PCR products were
further examined by the
ABI 3100 capillary
electrophoresis
analyzer
• ABI prism software
was used to interpret
data
Relative MSI Frequency in Various MMR Defective Lines
Relative frequency of shifts
30
25
20
15
10
5
0
Unique
wt avg.
MSH2-#1
RNAi
MLH1-
MLH3-
PMS2-
Funding of the Project
This work was supported by a grant from
the Howard Hughes Medical Institute
As well as in part by the Dr. John Hays’ lab
Acknowledgements
Dr. John Hays for acting as a mentor
throughout the research process
Adela Torres, Peter Hoffman, Stephanie
Bollmann, and Dr. Huixian Wang for
training and guidance